We separated the corticostriatal input into four major streams: prefrontal (insular and orbitofrontal cortices, as well as the frontal association area), motor (primary and secondary motor cortices), sensory (primary and secondary somatosensory cortices), and limbic (prelimbic, retrosplenial, cingulate, perirhinal, and entorhinal cortices). We found that while prefrontal cortical structures provided a similar proportion of input to direct- and indirect-pathway MSNs (21.3% ± 3.3% of total cortical input 5-FU cost versus

25.1% ± 2.4% onto D1R cells versus D2R cells, respectively; all reported values are mean ± 1 SEM, p = 0.4 by two-tailed t test), other cortical structures provided considerably biased synaptic input to one stream or the other (Figure 4G). Motor cortices provided significantly higher proportions of input to the indirect pathway (28.9% ± 3.3% versus 43.1% ± 3.2%, p = .007). In contrast, somatosensory and limbic cortices tended to provide a stronger proportion of input to the direct

pathway (somatosensory: 38.4% ± 3.6% versus 29.3% ± 2.6%, p = 0.05; limbic: 11.3% ± 3.4% versus 2.5% ± 1.2%, p = 0.02). As seen in Figure 3, biased sensory and motor input almost exclusively arose from the primary cortical structures, whereas all limbic structures appeared to provide a larger proportion of inputs to direct pathway MSNs. These data provide evidence for some segregation of cortical input to the two striatal projection PAK6 pathways. To further demonstrate the difference in the proportions of cortical input innervating the direct MAPK Inhibitor Library in vivo and indirect pathways, we performed a center of gravity analysis to determine the center of corticostriatal input to D1R and D2R MSNs (see Supplemental Experimental Procedures). Overall, corticostriatal inputs to the direct pathway were significantly posterior to the inputs to indirect pathway neurons (0.63 mm ± 0.11 mm rostral to bregma for D1R-Cre mice, 0.93 mm ± 0.06 mm for D2R-Cre mice, p = 0.03 by two-tailed t test). One D1R-Cre mouse with considerable prefrontal input had significantly shifted center of gravity compared to all other animals (p <

0.05 via Grubbs’ outlier test) and was removed from visual comparison (with outlier removed, center of gravity was 0.54 mm ± 0.07 mm for D1R-Cre mice, versus 0.93 mm ± 0.06 mm for D2R-Cre mice, p = 7 × 10−4 by two-tailed t test, Figure 4H; outlier is indicated by faded circle). The dashed line delineates the border between primary somatosensory and primary motor cortex at the sagittal slice containing both cohorts’ center of gravity (2.04 mm lateral from the midline). Both the lateral-medial and dorsal-ventral center of gravity positions were nearly identical between D1R-Cre and D2R-Cre mice (LM: 2.08 mm ± 0.10 mm lateral from the midline versus 2.05 mm ± 0.07 mm for D1R-Cre versus D2R-Cre mice, DV: 2.07 mm ± 0.05 mm deep from bregma versus 2.07 mm ± 0.06 mm).